Mitochondria-tau association promotes cognitive decline and hippocampal bioenergetic deficits during the aging.

Current studies indicate that pathological modifications of tau are associated with mitochondrial dysfunction, synaptic failure, and cognitive decline in neurological disorders and aging. We previously showed that caspase-3 cleaved tau, a relevant tau form in Alzheimer's disease (AD), affects mitochondrial bioenergetics, dynamics and synaptic plasticity by the opening of mitochondrial permeability transition pore (mPTP). Also, genetic ablation of tau promotes mitochondrial function boost and increased cognitive capacities in aging mice. However, the mechanisms and relevance of these alterations for the cognitive and mitochondrial abnormalities during aging, which is the primary risk factor for AD, has not been explored. Therefore, in this study we used aging C57BL/6 mice (2-15 and 28-month-old) to evaluate hippocampus-dependent cognitive performance and mitochondrial function. Behavioral tests revealed that aged mice (15 and 28-month-old) showed a reduced cognitive performance compared to young mice (2 month). Concomitantly, isolated hippocampal mitochondria of aged mice showed a significant decrease in bioenergetic-related functions including increases in reactive oxygen species (ROS), mitochondrial depolarization, ATP decreases, and calcium handling defects. Importantly, full-length and caspase-3 cleaved tau were preferentially present in mitochondrial fractions of 15 and 28-month-old mice. Also, aged mice (15 and 28-month-old) showed an increase in cyclophilin D (CypD), the principal regulator of mPTP opening, and a decrease in Opa-1 mitochondrial localization, indicating a possible defect in mitochondrial dynamics. Importantly, we corroborated these findings in immortalized cortical neurons expressing mitochondrial targeted full-length (GFP-T4-OMP25) and caspase-3 cleaved tau (GFP-T4C3-OMP25) which resulted in increased ROS levels and mitochondrial fragmentation, along with a decrease in Opa-1 protein expression. These results suggest that tau associates with mitochondria and this binding increases during aging. This connection may contribute to defects in mitochondrial bioenergetics and dynamics which later may conduce to cognitive decline present during aging.

Inorganic polyphosphate's role in energy production and mitochondrial permeability transition pore opening in tick mitochondria.

Inorganic polyphosphate (polyP) is a biopolymer composed of phosphoanhydride-linked orthophosphate molecules. PolyP is engaged in a variety of cellular functions, including mitochondrial metabolism. Here, we examined the effects of polyP on electron transport chain enzymes and F1 Fo ATP synthase in tick embryos during embryonic development. The study found that polyPs containing medium and long chains (polyP15 and polyP65 ) enhanced the activity of complex I, complex II, complex III, and F1 Fo ATP synthase, while short polyP chains (polyP3 ) had no effect. The study also examined the activity of exopolyphosphatases (PPX) in various energy-demand situations. PPX activity was stimulated when ADP concentrations are high, characterizing a low-energy context. When complexes I-III and F1 Fo ATP synthase inhibitors were added in energized mitochondria, PPX activity decreased, whereas the mitochondrial uncoupler FCCP had no impact on PPX activity. Additionally, the study investigated the effect of polyP on mitochondrial swelling, finding that polyP causes mitochondrial swelling by increasing calcium effects on the mitochondrial permeability transition pore. The findings presented here to increase our understanding of the function of polyP in mitochondrial metabolism and its relationship to mitochondrial permeability transition pore opening in an arthropod model.

Cyclosporine A (CsA) prevents synaptic impairment caused by truncated tau by caspase-3.

During Alzheimer's (AD), tau protein suffers from abnormal post-translational modifications, including cleaving by caspase-3. These tau forms affect synaptic plasticity contributing to the cognitive decline observed in the early stages of AD. In addition, caspase-3 cleaved tau (TauC3) impairs mitochondrial dynamics and organelles transport, which are both relevant processes for synapse. We recently showed that the absence of tau expression reverts age-associated cognitive and mitochondrial failure by blocking the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial complex involved in calcium regulation and apoptosis. Therefore, we studied the effects of TauC3 against the dendritic spine and synaptic vesicle formation and the possible role of mPTP in these alterations. We used mature hippocampal mice neurons to express a reporter protein (GFP, mCherry), coupled to full-length human tau protein (GFP-T4, mCherry-T4), and coupled to human tau protein cleaved at D421 by caspase-3 (GFP-T4C3, mCherry-T4C3) and synaptic elements were evaluated. Treatment with cyclosporine A (CsA), an immunosuppressive drug with inhibitory activity on mPTP, prevented ROS increase and mitochondrial depolarization induced by TauC3 in hippocampal neurons. These results were corroborated with immortalized cortical neurons in which ROS increase and ATP loss induced by this tau form were prevented by CsA. Interestingly, TauC3 expression significantly reduced dendritic spine density (filopodia type) and synaptic vesicle number in hippocampal neurons. Also, neurons transfected with TauC3 showed a significant accumulation of synaptophysin protein in their soma. More importantly, all these synaptic alterations were prevented by CsA, suggesting an mPTP role in these negative changes derived from TauC3 expression.

Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca2+ Handling and Ca2+-Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion.

Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca2+ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca2+ (Ca2+ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca2+-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca2+ load and t1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury-thus favoring Ca2+ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle-which feed the mitochondrial complexes with the electrons removed from different substrates-as the result of accentuated stimulus of citrate synthase activity by BMMC.

Asymmetric Opening of Mitochondrial Permeability Transition Pore in Mouse Brain Hemispheres: A Link to the Mitochondrial Calcium Uniporter Complex

Abstract The prolonged entry of large amounts of calcium into the mitochondria through the mitochondrial calcium uniporter complex (MCUC) may cause the permeability transition pore (mPTP) to open, which contributes to the pathogenesis of several diseases. Tissue-specific differences in mPTP opening due to variable expression of MCUC components may contribute to disease outcomes. We designed this study to determine differential mPTP opening in mitochondria isolated from different regions of mouse brain and kidney and to compare it with the expression of MCUC components. mPTP opening was measured using mitochondria isolated from the left/right brain hemispheres (LH/RH, respectively) and from kidney cortex/medulla, while the expression level of MCUC components was assessed from total cellular RNA. Interestingly, LH mitochondria showed less calcium-induced mPTP opening as compared to RH mitochondria at two different calcium concentrations. Conversely, mPTP opening was similar in the renal cortex and renal medulla mitochondria. However, the kidney mitochondria demonstrated bigger and faster mPTP opening as compared to the brain mitochondria. Furthermore, asymmetric mPTP opening in the LH and RH mitochondria was not associated with the expression of MCUC components. In brief, this study demonstrates thus far unreported asymmetric mPTP opening in mouse brain hemispheres that is not associated with the mRNA levels of MCUC components.

Tamoxifen Sensitizes Acute Lymphoblastic Leukemia Cells to Cannabidiol by Targeting Cyclophilin-D and Altering Mitochondrial Ca2+ Homeostasis.

Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.

A systematic review of post-translational modifications in the mitochondrial permeability transition pore complex associated with cardiac diseases.

The mitochondrial permeability transition pore (mPTP) opening is involved in the pathophysiology of multiple cardiac diseases, such as ischemia/reperfusion injury and heart failure. A growing number of evidence provided by proteomic screening techniques has demonstrated the role of post-translational modifications (PTMs) in several key components of the pore in response to changes in the extra/intracellular environment and bioenergetic demand. This could lead to a fine, complex regulatory mechanism that, under pathological conditions, can shift the state of mitochondrial functions and, thus, the cell's fate. Understanding the complex relationship between these PTMs is still under investigation and can provide new, promising therapeutic targets and treatment approaches. This review, using a systematic review of the literature, presents the current knowledge on PTMs of the mPTP and their role in health and cardiac disease.

Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics.

Erythropoiesis is the most robust cellular differentiation and proliferation system, with a production of ∼2 × 1011 cells per day. In this fine-tuned process, the hematopoietic stem cells (HSCs) generate erythroid progenitors, which proliferate and mature into erythrocytes. During erythropoiesis, mitochondria are reprogrammed to drive the differentiation process before finally being eliminated by mitophagy. In erythropoiesis, mitochondrial dynamics (MtDy) are expected to be a key regulatory point that has not been described previously. We described that a specific MtDy pattern occurs in human erythropoiesis from EPO-induced human CD34+ cells, characterized predominantly by mitochondrial fusion at early stages followed by fission at late stages. The fusion protein MFN1 and the fission protein FIS1 are shown to play a key role in the progression of erythropoiesis. Fragmentation of the mitochondrial web by the overexpression of FIS1 (gain of fission) resulted in both the inhibition of hemoglobin biosynthesis and the arrest of erythroid differentiation, keeping cells in immature differentiation stages. These cells showed specific mitochondrial features as compared with control cells, such as an increase in round and large mitochondrial morphology, low mitochondrial membrane potential, a drop in the expression of the respiratory complexes II and IV and increased ROS. Interestingly, treatment with the mitochondrial permeability transition pore (mPTP) inhibitor, cyclosporin A, rescued mitochondrial morphology, hemoglobin biosynthesis and erythropoiesis. Studies presented in this work reveal MtDy as a hot spot in the control of erythroid differentiation, which might signal downstream for metabolic reprogramming through regulation of the mPTP.

New perspectives for hydrogen peroxide in the amastigogenesis of Trypanosoma cruzi in vitro.

Trypanosoma cruzi has a complex life cycle involving four life stages: the replicative epimastigotes and metacyclic trypomastigotes in the invertebrate host digestive tract, and intracellular amastigotes and bloodstream trypomastigotes in the mammalian host. Trypomastigotes can invade any nucleated cell, including macrophages, which produce ROS that enhance intracellular infection. However, how ROS modulate T. cruzi infection in the mammalian cell remains unclear. Therefore, the present work aimed to investigate the role of ROS during the stimulation of amastigogenesis in vitro. Our results showed that H2O2 improves the differentiation process in vitro and that it was impaired by Peg-Catalase. However, the antioxidants GSH and NAC had no influence on induced amastigogenesis, which suggests the specificity of H2O2 to increase intracellular differentiation. Amastigogenesis physiologically occurs in low pH, thus we investigated whether parasites are able to produce ROS during amastigogenesis. Interestingly, after 60 min of differentiation induction in vitro, we observed an increase in H2O2 production, which was inhibited by the mitochondrial-targeted antioxidant, mitoTEMPO and Cyclosporine A (a mitochondrial permeability transition pore -mPTP- inhibitor), suggesting mitochondrion as a H2O2 source. Indeed, quantitative real time (qPCR) showed an increase of the mitochondrial superoxide dismutase (FeSODA) gene expression after 60 min of induced amastigogenesis, reinforcing the hypothesis of mitochondrial ROS induction during intracellular differentiation of T. cruzi. The reduction of cellular respiration and the decreased ΔΨm observed during amastigogenesis can explain the increased mitochondrial ROS through mPTP opening. In conclusion, our results suggest that H2O2 is involved in the amastigogenesis of T. cruzi.

The Role of Mitochondrial Impairment in Alzheimer´s Disease Neurodegeneration: The Tau Connection.

Accumulative evidence has shown that mitochondrial dysfunction plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). Mitochondrial impairment actively contributes to the synaptic and cognitive failure that characterizes AD. The presence of soluble pathological forms of tau like hyperphosphorylated at Ser396 and Ser404 and cleaved at Asp421 by caspase 3, negatively impacts mitochondrial bioenergetics, transport, and morphology in neurons. These adverse effects against mitochondria health will contribute to the synaptic impairment and cognitive decline in AD. Current studies suggest that mitochondrial failure induced by pathological tau forms is likely the result of the opening of the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial mega-channel that is activated by increases in calcium and is associated with mitochondrial stress and apoptosis. This structure is composed of different proteins, where Ciclophilin D (CypD) is considered to be the primary mediator of mPTP activation. Also, new studies suggest that mPTP contributes to Aß pathology and oxidative stress in AD. Further, inhibition of mPTP through the reduction of CypD expression prevents cognitive and synaptic impairment in AD mouse models. More importantly, tau protein contributes to the physiological regulation of mitochondria through the opening/interaction with mPTP in hippocampal neurons. Therefore, in this paper, we will discuss evidence that suggests an important role of pathological forms of tau against mitochondrial health. Also, we will discuss the possible role of mPTP in the mitochondrial impairment produced by the presence of tau pathology and its impact on synaptic function present in AD.

Simple kinetic model of mitochondrial swelling in cardiac cells.

Mitochondria play an important role in both cell survival and cell death. In response to oxidative stress, they undergo opening of non-selective permeability transition pores (PTP) in the inner mitochondrial membrane. Sustained PTP opening triggers mitochondrial swelling due to increased colloidal osmotic pressure in the matrix accompanied by mitochondrial membrane depolarization and ATP hydrolysis. Mitochondrial swelling is the major factor leading to mitochondria-mediated cell death through both apoptosis and necrosis. Hence, precise estimation of the threshold parameters of the transition of reversible swelling to irreversible swelling is important for understanding the mechanisms of PTP-mediated cell death as well as for the development of new therapeutic approaches targeting the mitochondria under pathological conditions. In this study, we designed a simple kinetic model of the Ca2+ -induced mitochondrial swelling that describes the mechanisms of transition from reversible to irreversible swelling in cardiac mitochondria. Values of kinetic parameters calculated using parameter estimation techniques that fit experimental data of mitochondrial swelling with minimum average differences between the experimental data and model parameters. Overall, this study provides a kinetic model verified by data simulation and model fitting that adequately describes the dynamics of mitochondrial swelling.

Maternal intake of trans-unsaturated or interesterified fatty acids during pregnancy and lactation modifies mitochondrial bioenergetics in the liver of adult offspring in mice.

The quality of dietary lipids in the maternal diet can programme the offspring to diseases in later life. We investigated whether the maternal intake of palm oil or interesterified fat, substitutes for trans-unsaturated fatty acids (FA), induces metabolic changes in the adult offspring. During pregnancy and lactation, C57BL/6 female mice received normolipidic diets containing partially hydrogenated vegetable fat rich in trans-unsaturated fatty acids (TG), palm oil (PG), interesterified fat (IG) or soyabean oil (CG). After weaning, male offspring from all groups received the control diet until day 110. Plasma glucose and TAG and liver FA profiles were ascertained. Liver mitochondrial function was accessed with high-resolution respirometry by measuring VO2, fluorimetry for detection of hydrogen peroxide (H2O2) production and mitochondrial Ca2+ uptake. The results showed that the IG offspring presented a 20 % increase in plasma glucose and both the IG and TG offspring presented a 2- and 1·9-fold increase in TAG, respectively, when compared with CG offspring. Liver MUFA and PUFA contents decreased in the TG and IG offspring when compared with CG offspring. Liver MUFA content also decreased in the PG offspring. These modifications in FA composition possibly affected liver mitochondrial function, as respiration was impaired in the TG offspring and H2O2 production was higher in the IG offspring. In addition, mitochondrial Ca2+ retention capacity was reduced by approximately 40 and 55 % in the TG and IG offspring, respectively. In conclusion, maternal consumption of trans-unsaturated and interesterified fat affected offspring health by compromising mitochondrial bioenergetics and lipid metabolism in the liver.

Development or disease: duality of the mitochondrial permeability transition pore.

Mitochondria is not only a dynamic organelle that produces ATP, but is also an important contributor to cell functions in both development and cell death processes. These paradoxical functions of mitochondria are partially regulated by the mitochondrial permeability transition pore (mPTP), a high-conductance channel that can induce loss of mitochondrial membrane potential, impairment of cellular calcium homeostasis, oxidative stress, and a decrease in ATP production upon pathological activation. Interestingly, despite their different etiologies, several neurodegenerative diseases and heart ischemic injuries share mitochondrial dysfunction as a common element. Generally, mitochondrial impairment is triggered by calcium deregulation that could lead to mPTP opening and cell death. Several studies have shown that opening of the mPTP not only induces mitochondrial damage and cell death, but is also a physiological mechanism involved in different cellular functions. The mPTP participates in regular calcium-release mechanisms that are required for proper metabolic regulation; it is hypothesized that the transient opening of this structure could be the principal mediator of cardiac and brain development. The mPTP also plays a role in protecting against different brain and cardiac disorders in the elderly population. Therefore, the aim of this work was to discuss different studies that show this controversial characteristic of the mPTP; although mPTP is normally associated with several pathological events, new critical findings suggest its importance in mitochondrial function and cell development.

Compensatory role of the NBCn1 sodium/bicarbonate cotransporter on Ca2+-induced mitochondrial swelling in hypertrophic hearts.

NBC Na+/HCO3- cotransporter (NBCn1) and NHE1 Na+/H+ exchanger have been associated with cardiac disorders and recently located in coronary endothelial cells (CEC) and cardiomyocytes mitochondria, respectively. Mitochondrial NHE1 blockade delays permeability transition pore (MPTP) opening and reduces superoxide levels, two critical events exacerbated in cells of diseased hearts. Conversely, activation of NBCn1 prevented apoptosis in CEC subjected to ischemic stress. We characterized the role of the NHE1 and NBCn1 transporters in heart mitochondria from hypertrophic (SHR) and control (Wistar) rats. Expression of NHE1 was analyzed in left ventricular mitochondrial lysates (LVML), by immunoblots. NHE1 expression increased by ~40% in SHR compared to control (P < 0.05, n = 4). To examine NHE1-mediated Na+/H+ exchange activity in cardiac hypertrophy, mitochondria were loaded with BCECF-AM dye and the maximal rate of pHm change measured after the addition of 50 mM NaCl. SHR mitochondria had greater changes in pHm compared to Wistar, 0.10 ± 0.01 vs. 0.06 ± 0.01, respectively (P < 0.05, n = 5). In addition, mitochondrial suspensions from SHR and control myocardium were exposed to 200 µM CaCl2 to induce MPTP opening (light-scattering decrease, LSD) and swelling. Surprisingly, SHR rats showed smaller LSD and a reduction in mitochondrial swelling, 67 ± 10% (n = 15), compared to control, 100 ± 8% (n = 13). NBC inhibition with S0859 (1 µM) significantly increased swelling in both control 139 ± 10% (n = 8) and SHR 115 ± 10% (n = 4). Finally, NBCn1 Na+/HCO3- cotransporter increased by twofold its expression in SHR LVML, compared to normal (P < 0.05, n = 5). We conclude that increased NBCn1 activity may play a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+-induced MPTP opening and swelling.

Elucidating Mitochondrial Electron Transport Chain Supercomplexes in the Heart During Ischemia-Reperfusion.

AIMS: Mitochondrial supercomplexes (SCs) are the large supramolecular assembly of individual electron transport chain (ETC) complexes that apparently provide highly efficient ATP synthesis and reduce electron leakage and reactive oxygen species (ROS) production. Oxidative stress during cardiac ischemia-reperfusion (IR) can result in degradation of SCs through oxidation of cardiolipin (CL). Also, IR induces calcium overload and enhances reactive oxygen species (mitROS) in mitochondria that result in the opening of the nonselective permeability transition pores (PTP). The opening of the PTP further compromises cellular energetics and increases mitROS ultimately leading to cell death. Here, we examined the role of PTP-induced mitROS in disintegration of SCs during cardiac IR. The relationship between mitochondrial PTP, ROS, and SCs was investigated using Langendorff-perfused rat hearts subjected to global ischemia (25 min) followed by short-time (5 min) or long-time (60 min) reperfusion in the presence or absence of the PTP inhibitor, sanglifehrin A (SfA), and the mitochondrial targeted ROS and electron scavenger, XJB-5-131. Also, the effects of CL deficiency on SC degradation, PTP, and mitROS were investigated in tafazzin knockdown (TazKD) mice. RESULTS: Cardiac IR induced PTP opening and mitROS generation, inhibited by SfA. Percent distributions of SCs were significantly affected by IR, and the effects were dependent on the reperfusion time and reversed by SfA and XJB-5-131. TazKD mice demonstrated a 40% lower SC I + III+IV with reduced basal mitochondrial PTP, ROS, and ETC complex activity. Innovation and Conclusion: Sustained reperfusion after cardiac ischemia induces disintegration of mitochondrial SCs, and PTP-induced ROS presumably play a causal role in SC disassembly. Antioxid. Redox Signal. 27, 57-69.

Mitochondrial permeability transition pore (MPTP) desensitization increases sea urchin spermatozoa fertilization rate.

Mitochondrial permeability transition pore (MPTP) is a protein complex whose opening promotes an abrupt increase in mitochondrial inner membrane permeability. Calcium signaling pathways are described in gametes and are involved in the fertilization process. Although mitochondria may act as Ca(2+) store and have a fast calcium-releasing mechanism through MPTP, its contribution to fertilization remains unclear. The work aimed to investigate the MPTP phenomenon in sea urchin spermatozoa and its role on the fertilization. Several pharmacological tools were used to evaluate the MPTP's physiology. Our results demonstrated that MPTP occurs in male gametes in a Ca(2+) - and voltage-dependent manner and it is sensitive to cyclosporine A. Additionally, our data show that MPTP opening does not alter ROS generation in sperm cells. Inhibition of MPTP in spermatozoa strongly improved the fertilization rate, which may involve mechanisms that increase the spermatozoa lifespan. The present work is the first report of the presence of a voltage- and Ca(2+) -dependent MPTP in gametes of invertebrates and indicates MPTP opening as another evolutionary feature shared by sea urchins and mammals. Studies about MPTP in sea urchin male gametes may contribute to the elucidation of several mechanisms involved in sperm infertility.

Antineoplastic copper coordinated complexes (Casiopeinas) uncouple oxidative phosphorylation and induce mitochondrial permeability transition in cardiac mitochondria and cardiomyocytes.

Copper-based drugs, Casiopeinas (Cas), exhibit antiproliferative and antineoplastic activities in vitro and in vivo, respectively. Unfortunately, the clinical use of these novel chemotherapeutics could be limited by the development of dose-dependent cardiotoxicity. In addition, the molecular mechanisms underlying Cas cardiotoxicity and anticancer activity are not completely understood. Here, we explore the potential impact of Cas on the cardiac mitochondria energetics as the molecular mechanisms underlying Cas-induced cardiotoxicity. To explore the properties on mitochondrial metabolism, we determined Cas effects on respiration, membrane potential, membrane permeability, and redox state in isolated cardiac mitochondria. The effect of Cas on the mitochondrial membrane potential (Δψm) was also evaluated in isolated cardiomyocytes by confocal microscopy and flow cytometry. Cas IIIEa, IIgly, and IIIia predominately inhibited maximal NADH- and succinate-linked mitochondrial respiration, increased the state-4 respiration rate and reduced membrane potential, suggesting that Cas also act as mitochondrial uncouplers. Interestingly, cyclosporine A inhibited Cas-induced mitochondrial depolarization, suggesting the involvement of mitochondrial permeability transition pore (mPTP). Similarly to isolated mitochondria, in isolated cardiomyocytes, Cas treatment decreased the Δψm and cyclosporine A treatment prevented mitochondrial depolarization. The production of H2O2 increased in Cas-treated mitochondria, which might also increase the oxidation of mitochondrial proteins such as adenine nucleotide translocase. In accordance, an antioxidant scavenger (Tiron) significantly diminished Cas IIIia mitochondrial depolarization. Cas induces a prominent loss of membrane potential, associated with alterations in redox state, which increases mPTP opening, potentially due to thiol-dependent modifications of the pore, suggesting that direct or indirect inhibition of mPTP opening might reduce Cas-induced cardiotoxicity.

Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa.

STUDY QUESTION: Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? SUMMARY ANSWER: Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation. WHAT IS KNOWN ALREADY: Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied. STUDY DESIGN, SIZE, DURATION: Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation. PARTICIPANTS/MATERIALS, SETTING, METHODS: To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Decreased calcein fluorescence after treatment with ionomycin (P < 0.05) suggests the opening of pores in the sperm IMM and this was accompanied by ΔΨm dissipation, increased ROS production and DNA fragmentation. ROS production occurred prior to the decrease in ΔΨm. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out. WIDER IMPLICATIONS OF THE FINDINGS: We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa. STUDY FUNDING/COMPETING INTERESTS: This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.

The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium.

Mineralocorticoid receptor (MR) antagonists decrease morbidity and mortality in heart failure patients for whom oxidative stress is usual; however, the underlying mechanism for this protection is unclear. Since aldosterone stimulates reactive oxygen species (ROS) production in several tissues, we explored its effect and the intracellular pathway involved in the rat myocardium. Aldosterone dose-dependently increased O2(-) production in myocardial slices. At 10 nmol/L, aldosterone increased O2(-) to 165 ± 8.8% of control, an effect prevented not only by the MR antagonists eplerenone and spironolactone (107 ± 7.8 and 103 ± 5.3%, respectively) but also by AG1478 (105 ± 8.0%), antagonist of the EGF receptor (EGFR). Similar results were obtained by silencing MR expression through the direct intramyocardial injection of a lentivirus coding for a siRNA against the MR. The aldosterone effect on O2(-) production was mimicked by the mKATP channel opener diazoxide and blocked by preventing its opening with 5-HD and glibenclamide, implicating the mitochondria as the source of O2(-). Inhibiting the respiratory chain with rotenone or mitochondrial permeability transition (MPT) with cyclosporine A or bongkrekic acid also canceled aldosterone-induced O2(-) production. In addition, aldosterone effect depended on NADPH oxidase and phosphoinositide 3-kinase activation, as apocynin and wortmannin, respectively, inhibited it. EGF (0.1 µg/mL) similarly increased O2(-), although in this case MR antagonists had no effect, suggesting that EGFR transactivation occurred downstream from MR activation. Inhibition of mKATP channels, the respiratory chain, or MPT did not prevent Akt phosphorylation, supporting that it happened upstream of the mitochondria. Importantly, cardiomyocytes were confirmed as a source of aldosterone induced mitochondrial ROS production in experiments performed in isolated cardiac myocytes. These results allow us to speculate that the beneficial effects of MR antagonists in heart failure may be related to a decrease in oxidative stress.

Novel o-naphthoquinones induce apoptosis of EL-4 T lymphoma cells through the increase of reactive oxygen species.

Novel ß-lapachone analogs 2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ1), 2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ3) and 2-methyl-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ7), which have trypanocidal activity, were assayed for cytotoxic effects on murine EL-4 T lymphoma cells. The NQs inhibited the proliferation of EL-4 cells at concentrations above 1µM. Nuclear staining of the EL-4 cells revealed chromatin condensation and a nuclear morphology compatible with the induction of apoptosis. Flow cytometry assays with annexin V-FITC and propidium iodide confirmed the cell death by apoptosis. Using electron paramagnetic resonance (EPR), a semiquinone radical was detected in EL-4 cells treated with NQs. In addition, a decrease in the GSH level in parallel with reactive oxygen species (ROS) production was observed. Preincubation with n-acetyl-l-cysteine (NAC) was able to reverse the inhibitory effects of the NQs on cell proliferation, indicating that ROS generation is involved in NQ-induced apoptosis. In addition, the NQs induced a decrease in the mitochondrial membrane potential and increased the proteolytic activation of caspases 9 and 3 and the cleavage of Poly (ADP-Ribose) Polymerase (PARP). In conclusion, these results indicate that redox cycling is induced by the NQs in the EL-4 cell line, with the generation of ROS and other free radicals that could inhibit cellular proliferation as a result of the induction of the intrinsic apoptosis pathway.